Role of the liver X receptors in skin physiology: Putative pharmacological targets in human diseases

Accepted Manuscript Title: Role of the liver X receptors in skin physiology: putative pharmacological targets in human diseases. Authors: Zangb´ewend´e Guy Ouedraogo, Allan Fouache, Amalia Trousson, Silv`ere Baron, Jean-Marc A. Lobaccaro PII: DOI: Reference:

S0009-3084(16)30195-5 http://dx.doi.org/doi:10.1016/j.chemphyslip.2017.02.006 CPL 4529

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Chemistry and Physics of Lipids

Received date: Revised date: Accepted date:

4-1-2017 22-2-2017 22-2-2017

Please cite this article as: Ouedraogo, Zangb´ewend´e Guy, Fouache, Allan, Trousson, Amalia, Baron, Silv`ere, Lobaccaro, Jean-Marc A., Role of the liver X receptors in skin physiology: putative pharmacological targets in human diseases.Chemistry and Physics of Lipids http://dx.doi.org/10.1016/j.chemphyslip.2017.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Role of the liver X receptors in skin physiology: putative pharmacological targets in human diseases.

Zangbéwendé Guy OUEDRAOGOa,b, Allan FOUACHEa,b, Amalia TROUSSONa,b, Silvère BARONa,b*, Jean-Marc A. LOBACCAROa,b*

a Université Clermont Auvergne, GReD, CNRS UMR 6293, INSERM U1103, 28, place Henri Dunant, BP38, F63001, Clermont-Ferrand, France. b Centre de Recherche en Nutrition Humaine d’Auvergne, 58 Boulevard Montalembert, F63009 Clermont-Ferrand, France

* Corresponding authors at: Université Clermont Auvergne, GReD, 10 Avenue Blaise Pascal, Campus Universitaire des Cézeaux, CS60026, F-63178 Aubiere, France . Tel.:+33 473 40 74 16 (JMAL); fax:+33 473 40 70 42. E-mail addresses: [email protected] (S. Baron); [email protected] (J.-M.A. Lobaccaro).

Abstract Liver X receptors (LXRs) are members of the nuclear receptor superfamily that have been shown to regulate various physiological functions such as lipid metabolism and cholesterol homeostasis. Concordant reports have elicited the possibility to target them to cure many human diseases including arteriosclerosis, cancer, arthritis, and diabetes. The high relevance of modulating LXR activities to treat numerous skin diseases, mainly those with exacerbated inflammation processes, contrasts with the lack of approved therapeutic use. This review makes an assessment to sum up the findings regarding the physiological roles of LXRs in skin and help progress towards the therapeutic and safe management of their activities. It focuses on the possible pharmacological targeting of LXRs to cure or prevent selected skin diseases.

Abbreviations used: ABCA1: ATP-binding cassette 1; ABCA12: ATP-binding cassette 12; ABCG1: ATP Binding cassette subfamily G member 1; Apo: apolipoprotein; CAT: catalase; COX-2: cyclooxygenase-2; DR4: direct repeat 4; EGCG: epigallocatechin-3-gallate; ERK, extracellular signal-related

kinase;

FLG:

filaggrin;

GBA:

beta-glucocerebrosidase;

HETE:

Hydroxyeicosatetraenoic acid; HMGCoA: 3-hydroxy-3-methyl-glutaryl-coenzyme A; IFNG: interferon gamma; IL1A: interleukin 1 alpha; IL1B: interleukin 1 Beta; IL6: Interleukin 6; IL8: interleukin 8; iNOS: inducible nitric oxide synthase; IVL: involucrin; LOR: loricrin; LPCAT3: lysophosphatidylcholine acyltransferase 3; LPS: lipopolysaccharide; LXR: liver X receptor; LXRE: LXR response element; MCP-1: monocyte chemoattractant protein-1; MDC: macrophage-derived chemokine; MITF, microphtalia-associated transcription factor; MMP: matrix metallopeptidase; MYC: v-myc avian myelocytomatosis viral oncogene homolog; NADPH: reduced form of nicotinamide adenine dinucleotide phosphate (NADP+); PPARG: peroxisome proliferator activated receptor gamma; PTGES1: microsomal prostaglandin E synthase; RXR: retinoid X receptor; SLiMs: selective liver X receptor modulators; TGM1: transglutaminase 1; TNFA: tumor necrosis factor A; TRP, tyrosine-related protein; UV: ultraviolet ; VDR: vitamin D (1,25-dihydroxyvitamin D3) receptor ; VEGFA: vascular endothelial growth factor A.

Keywords: LXRs; skin diseases; Selective liver X receptor modulators SLiMs; oxysterols; pharmacological targets

1.

Introduction

1.1.Structure and functioning of the LXRs Liver X receptors are members of the nuclear receptor superfamily (Willy et al., 1995; Maqdasy et al., 2016). They were early identified as a stage managers of lipid (including cholesterol) metabolism as well as homeostasis (Tobin et al., 2002). Two highly similar receptors have been described, LXRα (NR1H3) and LXRβ (NR1H2), encoded by distinct genes located respectively on the human genomic regions 11p11.2 and 19q13.3. They harbor the common structure of the nuclear receptor superfamily, made of three organized domains: i) the amino-terminal domain is a regulatory domain of the transcriptional activity, which undergoes regulatory post-translational modifications; ii) the central DNA-binding domain allows both recognition and binding to the LXR response element (LXRE), a specific DNA sequences also called direct repeat 4 (DR4), that consists in direct repeats of 5’-AGGTCA-3’ separated by four nucleotides (Willy et al., 1995); iii) the carboxy-terminal region contains a characteristic hydrophobic pocket for ligand binding and interaction with co-activators and co-repressors. While sharing a high rate amino-acid identity (77%) in their ligand- and DNAbinding domains, the two human LXR proteins differ in their amino-terminal domain and in the hinge domain located between the DNA- and the ligand-binding regions (see Viennois et al., 2011 for an extensive description) (Viennois et al., 2011). LXRα or LXRβ heterodimerizes with one of the 9-cis retinoic acid retinoid X receptors (RXRs or NR2B1-3) (Peet et al., 1998). Once bound to their respective ligands, oxysterol or 9-cis retinoic acid, allosteric changes in the heterodimer trigger the release of corepressors, the recruitment of coactivators and subsequent target gene transcription activation (El-Hajjaji et al., 2011; Lobaccaro et al., 2001; Peet et al., 1998). LXRs were so called at their discovery in 1994 from rat liver (Apfel et al., 1994) because the endogenous ligands had not been identified yet. The later discovery of oxygenated cholesterol derivatives (oxysterols) (Janowski et al., 1996) as LXR endogenous ligands, ejected them from the group of “orphan” nuclear receptors to be classified as “adopted” nuclear receptors. After two decades of intensive research to understand their physiological roles, the study of

selective Lxr knockout mice as well as the testing of natural and synthetic ligands pointed out that LXRs may be “druggable” targets in many human diseases including metabolic disorders, immunological upset and/or reproductive disturbances (see Maqdasy et al., 2016 for review). Hence, both LXRs are now considered as true endocrine receptors (Maqdasy et al., 2016). However, the extent of their biological functions highlighted the possible side effects that may accompany the management of their activities to prevent or cure diseases. This also may provide an explanation to the adverse events observed with the highest doses of the first clinically tried LXR agonist (LXR-623)(Katz et al., 2009). This awareness redirected research to the look for selective LXR modulators (SLiMs, (Viennois et al., 2011)) to take full advantage of the therapeutic and protective effects of LXR agonists while avoiding foreseeable drawbacks. Skin diseases, mainly those involving exacerbated inflammatory processes, are possible future indications of drug candidate LXR ligands (Viennois et al., 2012; Zheng et al., 2016), provided that efficient and safe drug is identified. Furthermore, by the feasibility of easy topical use, skin offers several possibilities for the design of targeted therapy (dermal topical medication) with limited or no general adverse events (see below, chapter III). 1.2.Natural ligands of the LXRs Oxysterols are the first and well documented LXR endogenous agonists. They include 20(S)-, 24(S)-, 22(R)-, 24(R)-, 27-hydroxycholesterols, and 24,25-epoxycholesterol, (Björkhem, 2002; Hessvik et al., 2012; Huang, 2014; Janowski et al., 1996; Schroepfer, 2000; Viennois et al., 2012). In addition to oxysterols, some of their downstream metabolites, mainly cholestenoic acids with a 3β-hydroxy-5-ene structure, activate LXRα and LXRβ in neuronal cells (Theofilopoulos et al., 2014). The 6α-hydroxylated bile acids have also been identified as LXRα selective activators (Song et al., 2000). In addition, some other lipid metabolites such as 5α,6α-epoxycholesterol, 7-ketocholesterol-3-sulfate, polyunsaturated fatty acids, arachidonic acid, prostaglandin F2α and ursodeoxycholic acid have been shown to antagonize LXR activation (Berrodin et al., 2010; Lee et al., 2014; Ou et al., 2001; Song et al., 2001; Yoshikawa et al., 2002; Zhuang et al., 2013). Besides those endogenous ligands, many agonists from fungi and many antagonists from plants and fungi have been described (see Huang et al., 2014 for review). However this effect needs to be demonstrated in vivo. It is currently accepted that human LXRs are ubiquitously expressed and both LXRα and β are present in skin (Russell et al., 2007). Besides, oxysterols are also present in this tissue. 3β-

hydroxy-5α-cholest-8(14)-en-15-one has been described in rat skin and hair (Emmons et al., 1988); this compound inhibits cholesterol synthesis by regulating 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase activity (Swaminathan et al., 1992) and has been shown to be a partial agonist for both LXRs (Schmidt et al. 2006). Sulfate esters of cholesterol are abundantly found in keratinized tissues such as skin (Zellmer and Lasch, 1997). They are synthesized by sulfotransferases (Falany et al., 2006), mainly SUL2B1b. It has been suggested that cholesterol sulfates are important for skin integrity and adhesion of the various layers (Figure 1). They also act in keratinocyte differentiation by controlling accumulation of proteins involved in the skin barrier, and perturbations in their metabolism are associated ichthyoses (Elias et al., 1984; Epstein et al., 1984; Webster et al., 1978; Williams and Elias, 1981; Williams et al., 1987). Skin has also been described as a “true” steroidogenic organ (Slominky et al. 2014; Thiboutot et al. 2003). As it, potent LXR ligands which are intermediate in the conversion of cholesterol to pregnenolone by CYP11A1 such as 22(R)-, 25- and 27-hydroxycholesterols could be synthesized. 25-hydroxycholesterol could also be produced keratinocytes under by UV light during photoaging (Olivier et al. 2016). Interestingly, 6-ketocholestanol could also be found in skin. This sterol does not have any known activity on LXRs but could inhibit cholesterol epoxide hydrolase which has been involved in cancer progression (Silvente-Poirot and Poirot, 2012. Altogether, LXRs are ligand-activated transcription factors; because plant-, fungi- and see animal-origin molecules could modulate LXR activities, identifying and developing medical, nutraceutical or cosmetic LXR modulators is a reasonable challenge. 2.

Roles of LXRs in skin physiology

The skin is an organ that plays, as a selective barrier, a central role in body protection and exchange with environment. This role is achieved thanks to a strict specialization of skin cells that build a well-structured histological architecture, which prevents invasion by pathogens and also controls flows of chemical and physical agents. The skin is made up of three distinct cellular layers (Figure 1): the inner, hypodermis, the dermis over the hypodermis, and the outermost, epidermis overlaying the dermis. Under physiologic conditions, the epidermal barrier is continuously maintained by intra-organ trafficking of contents as well as cell proliferation, migration and differentiation, from the stratum basale that provides autorenewal stem cells. LXR interferes with each of those processes as attested by in vitro and in vivo studies (see below).

2.1.LXRs inhibit proliferation and induce differentiation of skin cells Keratinocytes are the predominating cells of the skin. From the stratum basale, they undergo the vertical differentiation program to successively give rise to the stratum spinosum, the stratum granulosum, and finally and outermost to the stratum corneum made of anuclear bodies full of proteins and called corneocytes. These cells are embedded in a lipid matrix to form the outer skin barrier. Differentiation of keratinocytes to form the stratum corneum is accompanied by production of particular proteins such as loricrin, involucrin and filaggrin. Oxysterols inhibit keratinocytes proliferation and simultaneously induce their differentiation by activating LXRβ (Hanley et al., 2000; Kömüves et al., 2002). They increase expression of loricrin, involucrin, filaggrin and transglutaminase-1, that are markers of keratinocyte differentiation (Hanley et al., 2000; Kömüves et al., 2002). The result is the formation of a cornified envelop. In utero oxysterols stimulate development of the barrier permeability in fetal rat skin (Hanley et al., 1999) and improve barrier homeostasis in adult mice (Kömüves et al., 2002). The molecular mechanisms involved are provided by i) epidermal cholesterol, fatty acid, and sphingolipid synthesis; ii) increase in formation, secretion, and post-secretory processing of lamellar bodies; iii) induction of activity of the lipid processing enzyme βglucocerebrosidase; iv) expression of ABCA12, a membrane transporter of glucosylceramides into lamellar bodies (Jiang et al., 2008; Man et al., 2006). Surprisingly, despite such antiproliferative effect of LXR activation described in vitro and in vivo, it has been shown that topical application of activators of LXRs can slow down the adverse effects of exogenous glucocorticoids on the epidermis, including decrease of both proliferation and differentiation in mice (Demerjian et al., 2009). The neural crest derived pigment cells, the melanocytes, play crucial role in skin by protecting cells from UV radiation. They synthetize melanin polymer in melanosome organelles that are transferred to keratinocytes (Lin and Fisher, 2007). Recent studies have pointed out a possible role of LXRα in the pathogenesis of vitiligo, a skin pigmentation disorder with loss of melanocytes. An abnormal LXRα hyperactivatity has been described in vitiligo perilesional melanocytes (Kumar et al., 2010). Interestingly, LXRα single nucleotide polymorphisms (rs11039155 and rs2279238) have been associated with susceptibility to develop vitiligo (Agarwal et al., 2016). Early explanation incriminates a decrease in cell proliferation, an induction of cell death and reduction of MMP1, MMP2 and MMP9 due to LXRα activation in melanocytes (Kumar et al., 2012). Further studies are needed to better define the role of LXRα as well as the level of the ligands in this disease.

Besides keratinocytes and melanocytes, other skin cells, including sebocytes, fibroblasts and adipocytes, undergo reduction of proliferation and increase in differentiation when LXR agonists are added (Russell et al., 2007). Vice-versa, (-)-epigallocatechin-3-gallate (EGCG) impaired proliferation, adipose differentiation and lipid accumulation of 3T3-L1 mouse embryo fibroblasts while it down-regulated LXRα (Moon et al., 2007). Thus, activation of LXRs could inhibit proliferation and stimulate differentiation of various skin cell types (Figure 2). 2.2.Skin barrier maintenance Integrity of the skin is required to perform physiologic functions and prevent infections and trans-epidermal loss of water. Lipid composition of the skin plays hence a critical role. The epidermis pool of lipids is mainly composed of cholesterol, fatty acids, and ceramides that account about 50% (van Smeden et al., 2014). Intrinsic and extrinsic (photo-) aging modifies corneocyte morphology. Likewise, a change in spatial distribution of sterol cholesterol sulfate, a membrane stabilizing lipid, and in the proportion of both lignoceric acid (C24:0) and hexacosanoic acid (C26:0) are observed during aging (Starr et al., 2016). UV-induced skin aging results in elevation of matrix metalloproteinases that degrade skin collagen (Fisher et al., 1997) and in hyperactivation of the MAP kinase pathway, mainly ERK, JNK and p38 (Kim et al., 2006). Intrinsic aging and photoaging are associated to disrupted skin barrier with sagging, laxity and dryness. In addition, many skin diseases such as ichthyoses, atopic dermatitis and psoriasis disrupt skin barrier (Schmuth et al., 2015). In ichthyoses, there is a primary dysfunction of skin barrier caused by i) corneocyte defect due to mutations in transglutaminase 1 (TGM1) (Oji et al., 2010; Schmuth et al., 2013); ii) defect in extracellular lipid matrix including impaired ceramide synthesis (Eckl et al., 2013; Radner et al., 2013), reduced cholesterol (Elias et al., 2014); iii) abnormal cell-to-cell adhesion (Hachem et al., 2006). In atopic dermatitis (Elias and Schmuth, 2009; Sandilands et al., 2007) and in psoriasis (Bergboer et al., 2012; de Cid et al., 2009), primary and secondary barrier dysfunctions (Schmuth et al., 2015) coexist. The secondary dysfunctions usually include inflammatory processes. Interestingly, activation of LXRs improves the repair of skin barrier in experimental models of aging and skin diseases (Chang et al., 2008; Kim et al., 2006; Man et al., 2006; Schmuth et al., 2004). Cholesterol is essential for cell membrane synthesis. Because LXRs regulate lipid synthesis and trafficking (Jiang et al., 2006, 2010), they strengthen the

relevance of their agonist ligands as drug candidates in injuries with disrupted skin barrier. Demonstration is provided by the study of mouse models. In acute barrier disruption, the need of cholesterol increases keratinocyte expression of ABCG1, a plasma membrane transporter involved in cholesterol efflux for the maintenance of lamellar body density, content, and secretion (Jiang et al., 2010). Furthermore, topical treatment of murine skin with LXR activators increases ABCG1 expression in murine epidermis, suggesting that it may help restoring skin barrier (Jiang et al., 2010). 2.3.LXRs modulate skin inflammation and wound healing Inflammation is a multistep biological response of body tissues to harmful stimulations. It involves immune cells, blood vessels, as well as molecular mediators and aims to rule out the cause, also to mop up damaged tissues and useless metabolites. It further induces tissue repair. Inflammation is a physiological process that is part of the innate immune system; it becomes pathological when it is exacerbated or long lasting (chronic) (Grivennikov et al., 2010; Hanahan and Weinberg, 2011; Karin et al., 2006). Acute inflammation chronologically follows three steps: i) an initiation or vascular phase; ii) an amplification or cellular phase; iii) a repair or resolving phase. The vascular phase involves vessel reaction to platelet- and resident cell secreted factors as well as plasma-derived factors. The amplification phase implicates attracted immune cells, mainly granulocytes responsible for phagocytosis and those involved in enzymatic granules release. Chronic inflammation involves, in its cellular phase, an implication of monocytes and lymphocytes. In each phase, inflamed tissues (resident cells and attracted cells) release mediators that modulate the process, such as proteins (chemokines such as TNF and interleukins), or lipids (eicosanoids such as prostaglandins, leukotrienes, thromboxanes and lipoxins, or resolvins). 2.4.LXR and prostaglandin synthesis Human cells synthetize lipids, depending on cell types and challenging stimuli. LXR agonists induce lipid accumulation in HaCaT keratinocytes by stimulating lipogenesis (Hong et al., 2010).

In

human

macrophages,

LXR

activation

induces

lysophosphatidylcholine

acyltransferase 3 (LPCAT3) that increases the pool of arachidonic acid to be mobilized from phospholipids for the production of eicosanoids (Ishibashi et al., 2013). It is thus not surprising that LXR activation may result in an exacerbation or conversely in a decrease of the inflammation process, depending on the produced eicosanoids. For instance, the release of prostaglandin E2, a pro-inflammatory mediator, is enhanced when macrophages are

preconditioned by LXR agonist (Ishibashi et al., 2013). Inversely, LXR agonists inhibit in cultured macrophages (Joseph et al., 2003) and in keratinocytes (Hong et al., 2010) the expression of COX-2, the major inducible enzyme involved in the synthesis of the prostaglandin intermediary product. LXR agonists furthermore downregulate inflammation in vivo (Joseph et al., 2003). Normal epidermal differentiation however needs subsequent synthesis of prostaglandins (sometimes proinflammatory) that are basally synthesized by COX-1 (Tiano et al., 2002). The putative relevance of using LXR agonists to offset COX-2 expression and decrease pro-inflammatory prostaglandin release should be assessed by taking into account their effect on COX-1 as for non-steroidal anti-inflammatory drugs. However, inhibition of COX-2 does not mean an anti-inflammatory effect as a matter of course. Actually, it has been shown that COX-2 has a pro-inflammatory effect by producing PGE2 in the early phase of inflammation in the rat carrageenan-induced pleurisy model of acute inflammation. However, COX-2 induces an anti-inflammatory profile of released prostaglandins with predominance of prostaglandin D2 and 15-deoxy-Δ12,14-prostaglandin J2 (Gilroy et al., 1999) at a later phase; this process is nowadays called “eicosanoid class switching”. Furthermore, evidence has been provided that the phospholipase A2/Cox2/prostaglandin E synthase/prostaglandin E2 axis activates this switching that results in the biosynthesis of lipoxins (Chandrasekharan and Sharma-Walia, 2015) and resolvins (Moro et al., 2016) that promote the resolution of the inflammatory process through various molecular and cellular processes (Mancini and Battista, 2011). In Alzheimer disease, prostaglandin F2α has been reported to selectively antagonize LXR/RXR and RXR/RXR dimers to accelerate the inflammatory response to β-amyloid (Zhuang et al., 2013). However, such finding has not been reported so far in skin. 2.5.LXRs and leukotriene synthesis Early studies showed that 5-, 12- and 15-lipoxygenases are active in normal human skin and hyperactivated in psoriatic skin (Duell et al., 1988). Inhibition of the leukotriene B4 (LTB4) and 5-lipoxygenase enzyme involved in its synthesis, is a therapeutic basis of drugs designed against asthma (Chauhan and Ducharme, 2012) as well as other diseases with exacerbated inflammation (Rao et al., 2010). In human atherosclerotic plaque, IL-4 induces expression of active 15-lipoxygenase that inhibits LXRα expression and activation, surprisingly resulting in an anti-inflammatory alternative macrophage M2 phenotype (Chinetti-Gbaguidi et al., 2011). How LXRα is inhibited by 15-lipoxygenase remains unclear. More explanation is provided by

the report that overexpression of arachidonate 5-lipoxygenase–activating protein (ALOX5AP or FLAP) in mouse adipose tissue leads to lipoxin A4 production and protects against dietinduced obesity, insulin resistance and inflammation (Elias et al., 2016). Analysis of lipoxin role in non-skin models reveales the anti-inflammatory properties of lipoxins that are lipid metabolites from arachidonic acid through lipoxygenases (Chandrasekharan and SharmaWalia, 2015). 2.6.LXRs and production of reactive oxygen species Treatment of lipopolysaccharides (LPS)-activated cells with endogenous or exogenous LXR activating ligands suppresses inducible nitric oxide (NO) synthase (iNOS) both at mRNA and protein levels, and thus subsequent NO production (Crisafulli et al., 2010; Hong et al., 2010; Joseph et al., 2003; Yasuda et al., 2005). This strengthened the early studies showing that LXR inhibition by 7-ketocholesterol resulted in an increased generation of reactive oxygen species by enhancing the expression of NADPH oxidase subunits in primary human macrophages (Pedruzzi et al., 2004) and in human U937 promonocytic leukemia (LemaireEwing et al., 2005). 2.7.LXRs inhibits pro-inflammatory cytokines production and release In skin diseases with exacerbated inflammation such as psoriasis, a low level of LXRα is found together with a high accumulation of pro-inflammatory IL-6, IL-8 and IFN-γ chemokines (Gupta et al., 2009). LXRs regulate pro-inflammatory cytokine synthesis and release depending from the presence/absence of an inflammatory stimulation. Treatment of unstimulated human peripheral blood monocytes or the differentiated macrophage cell line THP-1 with LXR and/or RXR agonists results in the specific induction of the potent proinflammatory cytokine TNFα (Landis et al., 2002). When the same cells are stimulated by LPS, they released IL-6 and IL-1β simultaneously with TNFα. However the release of cytokines when the cells are simultaneously treated with LPS and LXR/RXR ligands were not described by the authors. Besides, LXR ligands dampen the release of pro-inflammatory cytokines under inflammatory conditions. For instance, IL-1α and TNFα are downregulated in the oxysterol-treated sites from both irritant and allergic contact models of dermatitis compared to untreated sites (Fowler et al., 2003). Activation of LXRs also decreases TNFαactivated expression of pro-inflammatory IL-8, IL-6, and IL-1β and UV-induced IL-8 and TNFα (Chang et al., 2008).

Moreover, LXR activation by the agonist T0901317 has antifibrotic effects in bleomycininduced skin fibrosis by interfering with infiltration of macrophages and their release of the pro-fibrotic IL-6 (Beyer et al., 2015). In formaldehyde-induced pain mouse model, deletion of Lxrβ in mouse enhances the formalin-induced inflammation, with more activated microglia and astrocytes in the spinal cord and results in hyper reactivity to pain (Bao et al., 2016). The lack of Lxrβ was accompanied by elevated levels of pro-inflammatory cytokines IL-1β, TNF-α as well as NFκB in the formalin-injected paw. Altogether this suggests that LXRβ could also be a target for the development of analgesics. 2.8.LXRs decreases production and release of matrix metalloproteinases Matrix metalloproteinases (MMPs) are a family of Zn2+-containing endopeptidases which degrades extracellular matrix components. MMPs are involved in remodeling of tissues in physiological as well as pathological contexts, including metastasis and tissue infiltration by cancer cells, inflammation, organ development, angiogenesis, and wound healing (Brinckerhoff and Matrisian, 2002). Basal expression of MMP2 and MMP9 in LXRβknockout mice is higher than littermate controls, suggesting that LXRβ may repress MMPs (Chang et al., 2008). Furthermore, exposure to UV triggers overexpression of MMP2 and MMP9 in hairless mouse skin (Chang et al., 2008) and in human skin (Fisher et al., 1996), making a link between UV-induced skin inflammation (or aging) with MMPs. In addition, cultured fibroblasts displayed high levels of MMP3 and MMP13 (mouse orthologous of human MMP1). Moreover LXR activation by ligands significantly impaired the expression of these MMPs in wild type but not LXRβ knock out fibroblasts, thus confirming that LXR ligands mediated the negative regulation of MMP expression through LXRβ. Similar conclusions have been drawn from studies in other models (Castrillo et al., 2003; Laragione and Gulko, 2012). However, a study reported that MMP9 is not regulated in human monocyte-derived dendritic cells by LXR activation by T0901317 (Bruckner et al., 2012). 2.9.LXRs and angiogenesis LXRβ subsequently modulates signaling by ALK-1, the transforming growth factor beta (TGF-β) receptor family member (Mo et al., 2002) involved in hereditary hemorrhagic telangiectasia -Rendu-Osler-Weber syndrome- by mutation of its encoding gene ACVRL1 (Dupuis-Girod et al., 2010; Shovlin et al., 2000). LXRα and LXRβ transactivate the VEGFA promoter in macrophages by binding to an LXRE (Walczak et al., 2004). Furthermore, LXR

agonists have been shown to reverse high glucose-impaired endothelial progenitor cellmediated angiogenesis in vitro (Li et al., 2012), to promote angiogenesis in the ischemic brain and to improve mouse functional recovery after stroke (Chen et al., 2009; Cui et al., 2013). This is strengthened by the recent report that LXRα knockout mice display reduced angiogenesis, increased mortality and aggravation of heart failure after myocardial infarction (Liu et al., 2016). Some however reported that LXR activation inhibited endothelial sprouting and in vivo neo-angiogenesis probably due to disturbed VEGFR2 signaling, lipid raft localization, and cholesterol homeostasis (Noghero et al., 2012). This anti-angiogenic function of LXR may be beneficial to antitumor therapy (Pencheva et al., 2014) but requires further studies to decipher the exact mechanism, even though an early explanation has been provided through the transcriptional induction of tumor and stromal apolipoprotein-E (ApoE) (See above). 3.

Therapeutic relevance of LXRs in selected skin diseases

The discovery and development of SLiMs is a long and tedious process that needs several successive approaches based on molecular modeling, cell-based screening and toxicity studies on animal models. It is only after safety studies in healthy volunteers, registered in the ClinicalTrials.gov database, that the compounds could enter clinical trials (for a review on drug discovery of LXRs, see Hong and Tontonoz (Hong and Tontonoz, 2014). In Nature Reviews Drug Discovery 13,409 (2014), several patent applications were published related to LXRs by Anayaderm, Bristol-Myers Squibb, Exelixis, Vitae Pharmaceuticals, and Wyeth (Pfizer). A schematic representation of potential skin disorders that could be treated by specific LXR ligands is shown in Figure 3. 3.1.Psoriasis A significant decrease of LXRα expression has been described in psoriatic lesions compared to healthy skins (Mehta et al., 2013). Microarray analyses have shown that inflammatory chemokines, mainly monocyte chemoattractant protein-1 (MCP-1) and macrophage-derived chemokine (MDC), were elevated in patients compared to controls. Inflammatory chemokines were also highly expressed in affected vs. unaffected skin of patients with psoriasis (Mehta et al., 2013). In addition, MCP-1 and MDC were also higher in patient serum than in healthy subjects, while apolipoprotein-A1 (Apo-A1) was decreased in psoriatic patient serum in comparison to healthy subjects. For unknown reasons, despite such studies suggesting that

LXR activation may beneath to psoriatic patients (Gupta et al., 2009), no clinical trial of LXR based therapy has been made available for this disease so far. Moreover, an altered systemic metabolism of 12(S)-HETE has been reported in patients, resulting in increased urinary excretion of tetranor-12(S)-HETE with low level of its 12(S)HETE precursor in urine (Setkowicz et al., 2015). This suggests that systemic levels of 12(S)HETE are altered due to accelerated inactivation of this eicosanoid in beta-oxidation. Because 12-lipoxygenase is involved in the synthesis of those metabolites and the pro-resolving lipoxin A4 and lipoxin B4 (Chandrasekharan and Sharma-Walia, 2015), it could be speculated that a perturbation of this metabolic pathway may be the substratum of the exacerbated inflammation in psoriasis. Thus, normalizing the 12-lipoxygenase activity might be relevant for therapeutic strategy. 3.2.Atopic dermatitis Primary and secondary skin barrier dysfunctions coexist in atopic dermatitis (Schmuth et al., 2015), indicating that skin barrier restoration may be helpful in the therapeutic management of such diseases. In mouse model of dermatitis, transepidermal water loss was impaired by topical treatment with LXR or PPARγ agonists (Kim et al., 2012). Furthermore, murine atopic dermatitis has been reported to respond to topical LXR and PPARα and β/δ activators, resulting in normalized or improved epidermal hyperplasia and reduced histologic evidence of inflammation (Hatano et al., 2010). Normal lamellar membrane structures are subsequently restored by those treatments. Orally active LXR activators have been designed for the treatment of atopic dermatitis (Zheng et al., 2016). One of these, VTP 38543 developed by Vitae Pharmaeuticals as a topical treatment for eczema or atopic dermatitis, is expected to improve barrier function, to decrease inflammation in damaged skin tissue and to repair the damaged outer layer of skin by forming new cells and cementing the new cells together with secreted lipids. VTP-38543 is currently in a Phase 2a proof-of-concept clinical trial (ClinicalTrials.gov Identifier: NCT02655679) to assess the safety, tolerability and efficacy in healthy adult male and female patients with mild to moderate atopic dermatitis. 3.3.Post-surgery and post-radiotherapy wound healing Based on cellular and animal models, LXR activators may be efficient in improving postsurgery and post-radiotherapy wound healing, by normalizing skin homeostasis. Because of

their capacity to slow-down cell proliferation and induce cell differentiation, they should prevent hypertrophic scar or keloid, if that they are used at the late resolving phase. They should be avoided when cell proliferation is needed for re-epitheliation. Furthermore, their antifibrotic action (Beyer et al., 2015) may be a rational for their use during resolution phase. 3.4.Ichthyosis Existence of a primary skin barrier dysfunction strengthens the putative use of LXR agonists to repair and maintain skin barrier homeostasis (Schmuth et al., 2015). LXR agonists have been proven to be efficient in treating animal models of those diseases (Man et al., 2006). However because of genetic heterogeneity in ichthyoses involving genes such as TGM1 (Hennies et al., 1998), ABCA12, FLG and many others (for implicated genes and classification, see Oji et al, 2010 (Oji et al., 2010), it is possible that only some patients might respond to LXR activation. Other fundamental and preclinical studies on the role of LXRs in lipoxygenase pathways and in ichthyoses are required to better define the possible indications of LXR ligands in ichthyoses. 3.5.Photo-aging and sunburn Chang et al. have provided evidence that skin photo-aging, chronological aging and sunburn display molecular alterations and physical damages which are reproducible, for their major part, by LXRβ knockout in mice (Chang et al., 2008). They showed that LXR agonists may block COX-2 induction, pro-inflammatory chemokines and release of MMPs, and subsequently repair skin homeostasis and further inhibit skin thickening and wrinkle formation. The finding that aging dermal, but not epidermal, cells express more PTGES1 and COX-2 resulting in higher levels of prostaglandin E2 in elderly vs. young skin (Li et al., 2015) reinforces the relevance of using LXR agonists to slow down age- and UV-induced skin damages. 3.6.Skin cancer Melanoma is a cancer that schematically develops from the melanocytes, the pigmentcontaining cells. The primary cause of UV light exposure in people with low levels of skin pigments. It is mainly the most common type of malignancy in the Caucasian population and its incidence is dramatically increasing (Apalla et al. 2017). Interestingly, Lee et al. (Lee et al. 2013) demonstrated that natural (22(R)-hydroxycholesterol) and synthetic ligands of LXRs inhibit melanogenesis. This process goes through the decrease expression of tyrosine,

tyrosine-related protein 1 (TRP-1) and TRP-2, acceleration of degradation of microphtaliaassociated transcription factor (MITF), an important regulator melanogenesis, and the activation of extracellular signal-related kinase (ERK) and its downstream target ras, a small GTPase protein. More importantly LXRs activated by synthetic ligands could also block the proliferation of melanoma in cell culture (B16F10 and A-376) and in xenografts (Zhang et al. 2014). This occurs through the induction of apoptosis (caspase-3 cleavage). However, Pencheva et al. (Pencheva et al. 2014) also pointed out the reduction of ApoE, known to have suppressive effects on metastasis. These effects were mediated by LXRβ and not LXRα. Conversely to Zhang et al. (2014), the authors did not show any modification of proliferation or survival of melanoma cells. At last Pencheva’s work opens a new field of investigations for LXR ligands useful for melanoma as they have a clear significant effect of tumor growth of melanoma tumors resistant to dacarbazine (a clinically approved chemotherapeutic agent) and vemurafenib (a serine/threonine kinase B-Raf inhibitor).

4.

Conclusion and perspectives for LXR-based therapy in skin diseases

LXRs are promising pharmacological targets for several skin diseases as they transcriptional activity may be modulated by lipophilic molecules that could easily cross the skin structure. However some concerns should be addressed in the light of recent knowledges on the role of lipoxins and resolvins. Hence, it is important to know how LXRs influence the metabolism of polyunsaturated fatty acids by lipoxygenases. Furthermore, the design of clinical trials with LXR modulators should take into account the right selection of the targeted pathological process and also the moment of using LXR modulators in the evolution of the disease. Because LXRs play critical roles in several organs and physiological processes (Maqdasy et al., 2016), a cautious management of LXR modulators is undoubtedly necessary for adverse event prevention. Since LXRs are nuclear transcription factors presented as two proteins with specific cell expression, modulators should be adequately vectored (Katz et al., 2009). However, topical use in skin may help decreasing systemic side effects and also allow sufficient dose delivery to the targets. The next generation of SLiMs to be clinically tested should thus include the possibility of an external (topical) administration. They should also be able to access to their targets despite skin barrier permeability disruption that usually exists in the potential indicated diseases. The divergence in some research findings may be due to the complexity of the LXR regulated

pathways. Therefore, in vitro studies are useful to make a first screening for active drugs, and in vivo testing, for efficiency demonstration.

Conflict of interest: We have read and understood the Chemistry and Physics of lipids policy on declaration of interests and declare that Z. G. Ouedraogo is associate researcher funded by La Régie Municipale des Grands Thermes (La Bourboule, France) and Auvergne Region. The funders had no role in the redaction of this review, decision to publish, or preparation of the manuscript. Authors have no competing interest for the submitted review.

Funding: Part of this study is supported by specific grants from European Regional Development Fund (FEDER), Auvergne Region, La Régie Municipale des Grands Thermes (La Bourboule, France), GREENTECH SA (Saint-Beauzire, France).

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Hair

Stratum corneum Stratum lucidum Stratum granulosum

Epidermis

Keratinocyte Neutrophil

Stratum spinosum

CD4+ T Cell Melanocyte Merkel cell

Stratum basale

X Papillary layer

Fibroblast

Dermis Reticular layer

X Hypodermis (Subcutis)

Meissners corpuscle Sebaceous gland Arrector pili muscle Sweat gland

Paccinian corpuscle

Nerve Vein Artery Adipocyte

Macrophage Langerhans cell CD8+ T cell

Fig. 1. Schematic representation of human skin architecture. Skin is made of 3 layers that are from inner to outer, the hypodermis, the dermis and the epidermis. Epidermis is structured in 6 layers arising from proliferation and differentiation of keratinocytes from the stratum basale. Keratinocytes, fibroblasts, melanocytes and sebocytes the major skin resident cells while cells such as macrophages, dendritic cells, lymphocytes (CD4+or CD8+ T cells) and neutrophils are secondary attracted to the skin by released chemokines.

Cell proliferation Cell differentiation Skin barrier formation

p h y s i o l o g y

Co-activator

Oxysterol 9-cis-retinoate

L X R

R X R

Skin barrier repair Wound healing Inflammation

p a t h o l o g y

Fig. 2. Role of LXR in physiological and pathological processes. Through direct (transcriptional) or indirect (AP-1) regulation, LXR-RXR heterodimer modulates several targets involved in physiological or pathological conditions. LXR: liver X receptor, RXR: retinoid X receptor, DR4: direct repeat 4, LOR: loricrin, IVL: involucrin, FLG: filaggrin, TGM1: transglutaminase 1, ABCA1: ATP-binding cassette 1, ABCA12: ATPbinding cassette 12, ABCG1: ATP Binding cassette subfamily G member 1, VEGFA: vascular endothelial growth factor A, LPCAT3: lysophosphatidylcholine acyltransferase 3, COX-2: cyclo-oxygenase-2, iNOS: inducible nitric oxide synthase, IL1B: interleukin 1 Beta, IL6: Interleukin 6, IL8: interleukin 8, IFNG: interferon gamma, TNFA: tumor necrosis factor A, MMP2: matrix metallopeptidase 2, MMP9: matrix metallopeptidase 9.

ATOPIC DERMATITIS TNFA IL1A PSORIASIS

FLG GBA

FIBROTIC DISEASES

IFNG MYC IL8 IL6

PPARG CAT VDR

ICHTYOSES

TGM1 ABCA12 FLG LOR GBA

L X R

MELANOMA

ApoE

Caspase-3 cleavage

Fig 3. Putative therapeutic targets of LXR activators in selected skin diseases. In each of these skin diseases, perturbed (hyperactivated or repressed) pathways involving some LXR target genes have been associated to the pathogenesis or to the progression of the diseases. Those perturbations constitute rational for therapeutic development and use of SLiMs. LXR: liver X receptor, IFNG: interferon gamma, MYC: v-myc avian myelocytomatosis viral oncogene homolog, IL8: interleukin 8, PPARG: peroxisome proliferator activated receptor gamma, CAT:catalase, VDR: vitamin D (1,25- dihydroxyvitamin D3) receptor, TNFA: tumor necrosis factor A, IL1A: interleukin 1 alpha, FLG: filaggrin, GBA: beta-glucocerebrosidase, IL6: interleukin 6, TGM1: transglutaminase 1, ABCA12: ATP-binding cassette 12, LOR: loricrin; Apo E, apoliporotein E.